Sensitivity of Saccharomyces cerevisiae vegetative cells and spores to antimicrobial compounds

The sensitivity of Saccharomyces cerevisiae spores and vegetative cells to various antimicrobial compounds was compared. Sulphur dioxide, benzoic acid, potassium sorbate, salicylic acid, nystatin, actidione and pimaricin were tested. Generally, the Saccharomyces spores were more resistant than the corresponding vegetative cells. It was also observed that this greater resistance shown by the spores varied with the antimicrobial compound used. Only potassium sorbate was not selective and killed both vegetative cells and spores at about the same rate.     

Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

The mechanism by which potassium sorbate inhibits Bacillus cereus T and Clostridium botulinum 62A spore germination was investigated. Spores of B. cereus T were germinated at 35 degrees C in 0.08 M sodium-potassium phosphate buffers (pH 5.7 and 6.7) containing various germinants (L-alanine, L-alpha-NH2-n-butyric acid, and inosine) and potassium sorbate. Spores of C. botulinum 62A were germinated in the same buffers but with 10 mM L-lactic acid, 20 mM sodium bicarbonate, L-alanine or L-cysteine, and potassium sorbate. Spore germination was monitored by optical density measurements at 600 nm and phase-contrast microscopy. Inhibition of B. cereus T spore germination was observed when 3,900 micrograms of potassium sorbate per ml was added at various time intervals during the first 2 min of spore exposure to the pH 5.7 germination medium. C. botulinum 62A spore germination was inhibited when 5,200 micrograms of potassium sorbate per ml was added during the first 30 min of spore exposure to the pH 5.7 medium. Potassium sorbate inhibition of germination was reversible for both B. cereus T and C. botulinum 62A spores. Potassium sorbate inhibition of B. cereus T spore germination induced by L-alanine and L-alpha-NH2-n-butyric acid was shown to be competitive in nature. Potassium sorbate was also a competitive inhibitor of L-alanine- and L-cysteine-induced germination of C. botulinum 62A spores.     

Effects of potassium sorbate and other antibotulinal agents on germination and outgrowth of Clostridium botulinum type E spores in microcultures.

The effects of potassium sorbate, sodium hypophosphite, sodium tripolyphosphate, sodium nitrite, and linoleic acid on the germination and outgrowth of Clostridium botulinum type E spores were studied in microcultures. At pH 5.8 to 6.0 in liver veal agar, the germination rate was decreased to nearly zero with 1.0, 1.5, or 2.0% sorbate. At pH 7.0 t 7.2, these levels of sorbate afforded germination and outgrowth of abnormally shaped cells that were defective in cell division. At the high pH range, 0.5 or 1.0% hypophosphite had effects similar to those of sorbate. The use of 0.05% sodium nitrite with sorbate enhanced the lysis of outgrowing cells at pH 7.2 or lower. Emergence and elongation were inhibited by 0.05% linoleic acid with or without 1.0% sorbate at pH 7.0 to 7.2. The addition of 0.5% tripolyphosphate to media containing 1.5% sorbate at pH 7.1 prevented normal cell growth to an extent greater than with sorbate alone.     

Use of yeast spores as a biocatalyst

Vegetative cells of the yeast Saccharomyces cerevisiae 4011 efficiently sporulated at pH 7.7–8.0 in the presence of 1.0–3.0% of potassium acetate. Spores were prepared by lysing them with a lytic enzyme, zymolyase. Alkaline phosphatase (an enzyme selected as a model) in spores exhibited higher stability toward heat and pH than it did in vegetative cells, and was immobilized in a polyacrylamide gel lattice without any appreciable loss of activity. The activity of alkaline phosphatase in spores and immobilized spores was stably maintained during repeated use for the enzyme reactions. These results indicated the usefulness of yeast spores as a biocatalyst.     

The effect of hydroxycinnamic acids and potassium sorbate on the growth of 11 strains of spoilage yeasts

D. STEAD. 1995. Hydroxycinnamic acids and their derivatives occur widely in plants, fruits and wine. The effect of the common hydroxycinnamic acids (caffeic, coumaric and ferulic acids), at concentrations of 100 and 500 mg 1^-1, on growth of 11 strains of spoilage yeasts was measured spectrophotometrically and compared with that of potassium sorbate. Ferulic acid was the most generally inhibitory hydroxycinnamic acid. At 500 mg 1^-1 it appreciably inhibited Pichia anomala, Debaryomyces hansenii and Saccharomyces cerevisiae and prevented detectable growth of one strain each of P. anomala and D. hansenii. Caffeic acid was the least inhibitory compound and coumaric acid had an intermediate effect. The more resistant strains of yeast were P. membranaefaciens, Saccharomycodes ludwigii and Zygosaccharomyces bailii. Sensitivity to hydroxycinnamic acid was, in general, associated with sensitivity to potassium sorbate; at a given concentration potassium sorbate was more inhibitory than were any of the hydroxycinnamic acids.     

Sensitization of thermally injured spores of Bacillus stearothermophilus to sodium benzoate and potassium sorbate

The effects of sodium benzoate and potassium sorbate added to the recovery medium, at different pH values (6·5, 6·0 and 5·0), on the recovery rates and heat resistance of Bacillus stearothermophilus spores (ATCC 12980, 7953, 15951 and 15952) were investigated. Heated spores of strains 12980 and 7953 were inhibited by sorbate concentrations over 0·05%. Potassium sorbate at concentrations as low as 0·025%, and sodium benzoate at 0·1%, were very effective inhibitory agents for heat-damaged spores. Their effectiveness always increased at pH 5·0, at which no growth occurred, with sodium benzoate for strains 7953, 15951 and 15952, and with potassium sorbate for strains 15951 and 15952. Decimal reduction times, whenever recovery was possible, were not significantly ( P  > 0·05) affected. None of these compounds modified the z -values obtained for the spores of the four strains, which had a mean value of 7·53 ± 0·28.     

Procedure for mutagenizing spores of Saccharomyces cerevisiae

A procedure for inducing mutants of a homothallic strain of Saccharomyces cerevisiae is described. The essential parts of the procedure are long incubation in Glusulase, which preferentially kills vegetative cells instead of spores, and treatment in 9% ethyl methanesulfonate, which also preferentially kills vegetative cells instead of spores. Consequently, the viable population is virtually 100% spores.     

Resistance of yeast and bacterial spores to high voltage electric pulses

Spores of a yeast, Saccharomyces cerevisiae, and a bacterium, Bacillus subtilis, were exposed to high voltage electric pulses. The viabilities of spores and vegetative cells of the yeast were significantly decreased after the electric pulse treatment, and some of the spores and almost all of the cells were stained red with an agent, phloxine B. On the other hand, (endo) spores of the bacterium were highly resistant to the electric pulses and little decrease in viability was observed, although the viability of vegetative cells was sharply lowered. The results revealed marked structural and/or biochemical differences between eukaryotic and prokaryotic spores.     

Potential for Development of Tolerance by Penicillium digitatum and Penicillium italicum after Repeated Exposure to Potassium Sorbate

Two strains of Penicillium digitatum and one strain of Penicillium italicum were exposed to various levels of sorbic acid and potassium sorbate, and the MICs were determined. Selected strains of the molds were then repeatedly exposed to subinhibitory levels of the compounds to determine whether increased tolerance might develop. The MIC of sorbic acid (pH 4.75) to P. digitatum was between 0.02 and 0.025%. The MIC of sorbate (pH 5.5) to two strains of P. digitatum and P. italicum was found to be between 0.06 and 0.08%. Increasing levels of sorbate resulted in increasing growth suppression of the molds. Populations of P. digitatum were tested for increased tolerance to sorbic acid, and none was found. Individual molds that started from the same parent colony were examined for increased tolerance to potassium sorbate. Two P. digitatum strains developed no observable increased tolerance, but P. italicum developed a slight increase in tolerance to sorbate. When spores of P. italicum and P. digitatum were exposed to higher levels of sorbate for prolonged times, the fungicidal or fungistatic activity of the inhibitor was dependent upon pH, length of exposure time, level of sorbate, and the mold strain.     

Inactivation of vegetative cells,but not spores,of Bacillus anthracis,B. cereus,and B. subtilis on stainless steel surfaces coated with an antimicrobial silver- and zinc-containing zeolite formulation

Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25 degrees C and 80% relative humidity), the zeolite coating produced approximately 3 log(10) inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected.     

The effects of Korean propolis against foodborne pathogens and transmission electron microscopic examination

This study was performed to evaluate the effects of Korean propolis against foodborne pathogens and spores of Bacillus cereus and to investigate the antimicrobial activity against B. cereus structure by transmission electron microscopy (TEM). The antimicrobial effects of the Korean propolis were tested against foodborne pathogens including Gram-positive (B. cereus, Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (Salmonella typhimurium, Escherichia coli and Pseudomonas fluorescence) bacteria by agar diffusion assay. Gram-positive bacteria were more sensitive than were Gram-negative bacteria. The vegetative cells of B. cereus were the most sensitive among the pathogens tested with minimum inhibitory concentration (MIC) of 0.036 mg/μl of propolis on agar medium. Based on MIC, sensitivity of vegetative cells of B. cereus and its spores was tested in a nutrient broth with different concentrations of propolis at 37°C. In liquid broth, treatment with 1.8 mg/ml propolis showed bactericidal effect against B. cereus. B. cereus vegetative cells exposed to 7.2mg/ml of propolis lost their viability within 20 min. Against spores of B. cereus, propolis inhibited germination of spores up to 30 hours, compared to control at higher concentration than vegetative cells yet acted sporostatically. The bactericidal and sporostatic action of propolis were dependent on the concentration of propolis used and treatment time. Electron microscopic investigation of propolis-treated B. cereus revealed substantial structural damage at the cellular level and irreversible cell membrane rupture at a number of locations with the apparent leakage of intracellular contents. The antimicrobial effect of propolis in this study suggests potential use of propolis in foods.     

Effects of potassium sorbate on growth patterns, morphology, and heat resistance of Zygosaccharomyces rouxii at reduced water activity.

A study was made of the effects of potassium sorbate on growth, morphology, and heat sensitivity of an osmotolerant yeast, Zygosaccharomyces rouxii, grown in media (water activity (aw) 0.93) supplemented with glucose and sucrose. Growth patterns of Z. rouxii in YM broth supplemented with glucose (YMBG) and sucrose (YMBS) were similar, although increased potassium sorbate concentration in both media resulted in decreased growth rates. Growth in YMBS containing potassium sorbate was not as prolific as that in YMBG containing potassium sorbate. Inhibition of growth was indicated by decreased absorbance (at 600 nm) of cells grown in YMBS and in YMBG and YMBS supplemented with potassium sorbate at 600 or 1000 micrograms/mL. Slight decreases in cell size and alteration of cellular morphology were associated with increased potassium sorbate concentration. Plasmolysis increased as potassium sorbate concentration was elevated in YMBS but not in YMBG. Tolerance of Z. rouxii to potassium sorbate was enhanced by previous adaptation of cells in media with elevated potassium sorbate concentrations. Heat resistance of cells unadapted to potassium sorbate showed little or no increase regardless of culture age, but increased substantially in cells grown in media containing potassium sorbate, particularly YMBS.     

Effect of sorbic acid and potassium sorbate on growth ofBacillus cereus andBacillus subtilis in rice filling of Karelian pasty

Summary The Karelian pasty is a perishable bakery product: the pH of the rice filling is approximately 6.60 and the a_W=0.98. Contamination after baking was shown to be the most important reason for microbial spoilage. The rice fillings of the pasties were artificially contaminated by spores ofBacillus cereus andB. subtilis, and growth at room temperature was followed.B. cereus was totally inhibited by an addition of 0.20% sorbic acid or 0.40% potassium sorbate.B. subtilis was totally inhibited by an addition of 0.10% sorbic acid or 0.26% potassium sorbate. An addition of 0.10% sorbic acid to the filling before baking is recommended. This level of addition does not alter the organoleptic quality of the pasty but is a considerable safety factor for Karelian pasty production.     

Thermal destruction of dried vegetative yeast cells and dried bacterial spores in a convective hot air flow: strong influence of initial water activity

Thermal treatment of Bacillus subtilis spores and Saccharomyces cerevisiae cells dried on glass beads was performed at various initial water activities (in the range 0.10-0.90). Experiments were carried out at 150 degrees C, 200 degrees C and 250 degrees C for 5-120 s. Significant destruction of up to 10(7) vegetative cells and up to 10(5) spores g(-1) was achieved, depending upon treatment conditions. This study demonstrated that the initial water activity (a(w)) value of a sample is very important in the destruction or survival of microorganisms treated with hot air stresses. As described previously, the heat resistance of spores and vegetative cells was strongly enhanced by low initial a(w) values until an optimal a(w) value between 0.30 and 0.50, with maximal viability at 0.35 for both S. cerevisiae and B. subtilis. However, our results highlighted for the first time that very low initial a(w) values (close to 0.10) greatly improved the destruction of spores and vegetative cells. Factors and possible mechanisms involved in the death of vegetative cells and spores are discussed.     

Distinctness of spore and vegetative cellular fatty acid profiles of some aerobic endospore-forming bacilli

A gas chromatographic analysis method was employed to determine the cellular fatty acid (CFA) profiles of spores and vegetative cells of some aerobic endospore-forming bacilli. The harvests of experimental strains were processed to obtain pure spores and acquire whole cell fatty acid methyl esters for the subsequent gas chromatographic analysis, and the corresponding vegetative cells were set as control. Evaluation of reproducibility of spore CFA components revealed that, provided under standardized experimental procedure, spore CFA composition was stable enough for research purposes. Fatty acids recovered in spores in greater quantities were saturated branched-chain acids containing 15 and 17 carbon atoms, similar to the vegetative cells. Commonly, the proportions of saturated branched-chain acids in spores were greater than in vegetative cells. The dendrograms obtained by cluster analysis provided some meaningful taxonomic information of the experimental strains. The fatty acids analysis of spores seems to be a promising supplementary tool for the chemotaxonomic research of aerobic endospore-forming bacilli.     

Repair of Radiation Damage to Deoxyribonucleic Acid in Germinating Spores of Bacillus subtilis

The repair of deoxyribonucleic acid (DNA) in germinating spores was studied in comparison with that in vegetative cells. Radiation-induced single-strand breaks in the DNA of spores and of vegetative cells of Bacillus subtilis were rejoined during postirradiation incubation. The molecular weight of single-stranded DNA was restored to the level of nonirradiated cells. The rate of the rejoining of DNA strand breaks in irradiated spores was essentially equal to that in irradiated vegetative cells. The rejoining in spores germinating in nutrient medium occurred in the absence of detectable DNA synthesis. In this state, normal DNA synthesis was not initiated. Very little DNA degradation occurred during the rejoining process. On the other hand, in vegetative cells the rejoining process was accompanied by a relatively large amount of DNA synthesis and DNA degradation in nutrient medium. The rejoining occurred in phosphate buffer in vegetative cells but not in spores in which germination was not induced. Chloramphenicol did not interfere with the rejoining process in either germinating spores or vegetative cells, indicating that the rejoining takes place in the absence of de novo synthesis of repair enzyme. In the radiation-sensitive strain uvs-80, the capacity for rejoining radiation-induced strand breaks was reduced both in spores and in vegetative cells, suggesting that the rejoining mechanism of germinating spores is not specific to the germination process.     

Free proline content and sensitivity to desiccation and heat during yeast sporulation and spore germination.

Ascospores of a strain of Saccharomyces cerevisiae Hansen were less sensitive to desiccation and heat than vegetative cells. Desiccation resistance was acquired earlier during sporulation and lost later during spore germination than heat resistance. As spores matured, resistance to both stresses increased. With the exception of the first few hours in sporulation medium, when proline appeared to be utilized, the intracellular free proline content increased during sporulation and decreased during spore germination. Not all the proline lost could be detected in the germination medium, indicating that some was metabolically utilized by the germinating spores. Since exogenous proline supplied to vegetative or sporulating cells before desiccation increased their survival, it is suggested that the high level of free proline in mature spores may protect against desiccation stress.     

Enzymes and germination of spores of the yeast Saccharomyces cerevisiae

Enzymes in spores of the yeast Saccharomyces cerevisiae were determined and compared with those in vegetative cells. The activities of phosphatase, oxidase and enzymes in the glycolytic bypass were high (about 3–10 fold) in spores, whereas those of dipeptidase, protease, DNase, RNase and TCA cycle enzymes were low in spores, being only 50% of the activities in vegetative cells. Enzymes in the glycolysis, glutathione and acetate metabolic pathways remained unchanged before and after sporulation. Germination of the yeast spores was repressed in the presence of d-aspartate, d-glutamate, d,l-methionine, d,l-cysteine, l-isoleucine, l-histidine and l-threonine, or bivalent metal ions such as Ni²⁺, Co²⁺ and Zn²⁺.     

Glycolaldehyde Dehydrogenase,Its Involvement in Vitamin B6 Biosynthetic Pathway of Escherichia coli B

The deoxyribonucleic acid (DNA) polymerases were partially purified from spores and vegetative cells of Bacillus subtilis. Some biochemical properties of the enzymes from the spores were studied in comparison with those from the vegetative cells. The spores and vegetative cells had at least three species of DNA polymerases (DNA polymerase I, II and III). These DNA polymerases in spores could not be distinguished from those in vegetative cells, respectively, with regard to the reresponses to ionic strength, the sensitivity to thiol-blocking agents, the template specificity, pH and temperature optima in assay, and the sedimentation behavior. It is inferred that DNA polymerases from spores was essentially identical to those from vegetative cells.

The DNA polymerase activity decreased rapidly in the course of sporulation, and only about 20% is recovered in the spores, suggesting that an extentive inactivation mechanism of the enzymes would be involved during sporulation.     

Comparisons of Cells, Refractile Bodies, and Spores of Bacillus popilliae

Spores of Bacillus popilliae from infected larvae and refractile bodies produced in a Trypticase-barbiturate medium were similar but distinct from vegetative cells of this organism in protein, nucleic acid, and enzyme composition. The spores and refractile bodies were found to have catalase activity, some of which was heat-resistant. This enzyme was not found in the vegetative cells. The spores contained dipicolinic acid, but the refractile bodies did not. The latter were similar to cells in having considerably higher levels of phosphate extractable with cold trichloroacetic acid and of poly-beta-hydroxybutyrate than had the spores. Electron microscopy demonstrated conclusively that the refractile bodies are distinctly different from either cells or spores of B. popilliae. The possibility that these bodies are formed as a result of an aborted sporulation process is discussed.